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Accelerating wound healing
<-‘ T\ Scrambled
eggs and toasted
Genome integrity insurance: a potential role for Cadd45
nuclei
NEWMEVER, D. D., FARSCHON, D. M. and REED, 1. C. (1994) Cell-free apoptosis in Xenopus egg extracts: inhibition by Bcl-2 and requirement for an organelle fraction enriched in mitochondria Cell 79, 353-364 Perhaps the most useful tool for understanding a biological process is a cell-free system. One can fractionate it to identify the activities involved, use it to test compounds for inhibitory effects without worrying about their cytotoxicity, and ultimately reconstitute the process using minimal components. Hence, the significance of this report of a cell-free system for apoptosis. The hallmarks of apoptosis-membrane blebbing, chromatin condensation, nuclear disintegration and DNA fragmentation can be faithfully reproduced in Xenopus egg extracts. The longer the egg extract was pm-incubated at room temperature, the faster apoptosis happened when nuclei were added. The authors concluded that there is a ‘latent phase’, during which cytoplasmic reactions occur in preparation for subsequent destructive nuclear events. This is supported by the fact that 8~1-2 (an oncoprotein that delays or prevents apoptosis in viva) blocked the in vitro process only when added during the latent phase. Thus, the nucleus seems to have a more passive role in apoptosis than previously assumed. The effects of various pharmacological agents on apoptosis in this system were assessed for insight into the activities involved in the process. These studies implicated cysteine proteases, CTP-binding proteins and protein tyrosine phosphatases (or phosphotyrosine-dependent protein-protein interactions) in the apoptotic events. A crude fraction containing mitochondria and other dense organelles was required for apoptosis in the extract. What role mitochondria play is unknown, but future studies using the cell-free system should shed light on this and other questions.
Accelerating VOGT,
Hatton, ChongYee Khoo, Carl Smythe, Debbie Sweet and Steven Theg.
58
healing
P. M. et al. (1994) Genetically modified keratinocytes wounds reconstitute the epidermis Proc. Not/ Acad. SC;. USA 91, 9307-9311
transplanted
to
Penetrative injury, burning, scalding and ulceration of human skin pose a serious threat to health. Factors affecting the natural wound-healing process are the focus of current studies aimed at improving treatment of skin damage. In this paper, Vogt al. reportthe rapid colonization of deep cutaneous porcine wounds by transplanted epidermal-derived keratinocytes. The keratinocytes were first tagged by transfection with retroviral vectors encoding B-galactosidase to allow the subsequent positioning and survival of the cells to be determined by immunostaining of biopsies. A novel chamber system was placed over the wound to create a standardized environment. Significantly, restoration of epithelial integrity, assayed by the exclusion of protein from wound fluid, occurred several days earlier in keratinocyte-transplanted wounds compared with non-transplanted controls, demonstrating that healing had been accelerated. Furthermore, the authors showed the potential for genetically modifying the wound environment by successful transplantation of keratinocytes engineered to produce human growth hormone, which was detected for ten days in wound fluid. These results suggest that further application of this chamber technique will lead to a greater understanding of factors influencing wound healing, and they raise the hope that temporary expression of appropriate transgenes, such as growth factor genes, in transplanted epidermal cells may result in successful treatment of wounded tissue by gene therapy.
et
This month’s headlines were contributed by Donald Cullberg, David
wound
et
SMITH, M. L. al. (1994) Interaction of the p53-regulated protein Cadd45 with proliferating cell nuclear antigen Science 266, 1376-l 380 An important cellular response to DNA damage is the arrest of cell cycle progression at Cl and C2 checkpoints, which allows cells time to repair DNA before S phase or mitosis. The ~53 tumour suppressor protein is required for the Cl checkpoint in mammalian cells. Following DNA damage, ~53 induces expression of genes such as WAFT/C/PI and CADD45. p21 WAF”C’P1 is known to block cell cycle progression by inhibiting cyclin-dependent kinases (CDKs) and the replicationpromoting ability of proliferating cell nuclear antigen (PCNA), but the role of Gadd45 action has been unclear. Smith et al. show here that Cadd45 both promotes DNA repair and inhibits cell cycle progression, apparently through interactions with PCNA. Cadd45 coimmunoprecipitated with PCNA from cells treated with ionizing radiation or UV light to induce DNA damage. The interaction appears to be direct because bacterially produced and purified proteins also associated, albeit poorly, in Interestingly, excision repair in which also requires PCNA, was activated by addition of recombinant Gadd45 and inhibited following immunodepletion of endogenous Gadd45 from the cell-free extract. Furthermore, expression of CADD45 antisense RNA in ceils reduced survival following UV radiation. A role for Cadd45 in suppressing cell cycle progression was confirmed by transient transfection of GADD45 into cells, which inhibited DNA synthesis. Unlike p21 WAF’~C’P’, however, Gadd45 does not appear to bind directly to CDKs. The authors suggest that sequestration of PCNA into repair complexes by Gadd45 may both promote repair and inhibit PCNA action in replication.
vitro. vitro,
TRENDS IN CELL BIOLOGY VOL. 5 FEBRUARY 1995
eggs and toasted
Genome integrity insurance: a potential role for Cadd45
nuclei
NEWMEVER, D. D., FARSCHON, D. M. and REED, 1. C. (1994) Cell-free apoptosis in Xenopus egg extracts: inhibition by Bcl-2 and requirement for an organelle fraction enriched in mitochondria Cell 79, 353-364 Perhaps the most useful tool for understanding a biological process is a cell-free system. One can fractionate it to identify the activities involved, use it to test compounds for inhibitory effects without worrying about their cytotoxicity, and ultimately reconstitute the process using minimal components. Hence, the significance of this report of a cell-free system for apoptosis. The hallmarks of apoptosis-membrane blebbing, chromatin condensation, nuclear disintegration and DNA fragmentation can be faithfully reproduced in Xenopus egg extracts. The longer the egg extract was pm-incubated at room temperature, the faster apoptosis happened when nuclei were added. The authors concluded that there is a ‘latent phase’, during which cytoplasmic reactions occur in preparation for subsequent destructive nuclear events. This is supported by the fact that 8~1-2 (an oncoprotein that delays or prevents apoptosis in viva) blocked the in vitro process only when added during the latent phase. Thus, the nucleus seems to have a more passive role in apoptosis than previously assumed. The effects of various pharmacological agents on apoptosis in this system were assessed for insight into the activities involved in the process. These studies implicated cysteine proteases, CTP-binding proteins and protein tyrosine phosphatases (or phosphotyrosine-dependent protein-protein interactions) in the apoptotic events. A crude fraction containing mitochondria and other dense organelles was required for apoptosis in the extract. What role mitochondria play is unknown, but future studies using the cell-free system should shed light on this and other questions.
Accelerating VOGT,
Hatton, ChongYee Khoo, Carl Smythe, Debbie Sweet and Steven Theg.
58
healing
P. M. et al. (1994) Genetically modified keratinocytes wounds reconstitute the epidermis Proc. Not/ Acad. SC;. USA 91, 9307-9311
transplanted
to
Penetrative injury, burning, scalding and ulceration of human skin pose a serious threat to health. Factors affecting the natural wound-healing process are the focus of current studies aimed at improving treatment of skin damage. In this paper, Vogt al. reportthe rapid colonization of deep cutaneous porcine wounds by transplanted epidermal-derived keratinocytes. The keratinocytes were first tagged by transfection with retroviral vectors encoding B-galactosidase to allow the subsequent positioning and survival of the cells to be determined by immunostaining of biopsies. A novel chamber system was placed over the wound to create a standardized environment. Significantly, restoration of epithelial integrity, assayed by the exclusion of protein from wound fluid, occurred several days earlier in keratinocyte-transplanted wounds compared with non-transplanted controls, demonstrating that healing had been accelerated. Furthermore, the authors showed the potential for genetically modifying the wound environment by successful transplantation of keratinocytes engineered to produce human growth hormone, which was detected for ten days in wound fluid. These results suggest that further application of this chamber technique will lead to a greater understanding of factors influencing wound healing, and they raise the hope that temporary expression of appropriate transgenes, such as growth factor genes, in transplanted epidermal cells may result in successful treatment of wounded tissue by gene therapy.
et
This month’s headlines were contributed by Donald Cullberg, David
wound
et
SMITH, M. L. al. (1994) Interaction of the p53-regulated protein Cadd45 with proliferating cell nuclear antigen Science 266, 1376-l 380 An important cellular response to DNA damage is the arrest of cell cycle progression at Cl and C2 checkpoints, which allows cells time to repair DNA before S phase or mitosis. The ~53 tumour suppressor protein is required for the Cl checkpoint in mammalian cells. Following DNA damage, ~53 induces expression of genes such as WAFT/C/PI and CADD45. p21 WAF”C’P1 is known to block cell cycle progression by inhibiting cyclin-dependent kinases (CDKs) and the replicationpromoting ability of proliferating cell nuclear antigen (PCNA), but the role of Gadd45 action has been unclear. Smith et al. show here that Cadd45 both promotes DNA repair and inhibits cell cycle progression, apparently through interactions with PCNA. Cadd45 coimmunoprecipitated with PCNA from cells treated with ionizing radiation or UV light to induce DNA damage. The interaction appears to be direct because bacterially produced and purified proteins also associated, albeit poorly, in Interestingly, excision repair in which also requires PCNA, was activated by addition of recombinant Gadd45 and inhibited following immunodepletion of endogenous Gadd45 from the cell-free extract. Furthermore, expression of CADD45 antisense RNA in ceils reduced survival following UV radiation. A role for Cadd45 in suppressing cell cycle progression was confirmed by transient transfection of GADD45 into cells, which inhibited DNA synthesis. Unlike p21 WAF’~C’P’, however, Gadd45 does not appear to bind directly to CDKs. The authors suggest that sequestration of PCNA into repair complexes by Gadd45 may both promote repair and inhibit PCNA action in replication.
vitro. vitro,
TRENDS IN CELL BIOLOGY VOL. 5 FEBRUARY 1995